Radiation therapy methods

ABSTRACT

The present invention provides methods and kits for mitigating radiation induced tissue damage, improving the effectiveness of radiation therapy, to support bone marrow transplantation, and promoting megakaryocyte production and mobilization and platelet production, each method comprising the administration of an effective amount of angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, angiotensin II (AII), AII analogues, AII fragments or analogues thereof or AII AT 2  type 2 receptor agonists.

CROSS REFERENCE

This application is a Continuation of U.S. patent application Ser. No.10/341,001 filed Jan. 13, 2003, which is a Continuation of U.S. patentapplication Ser. No. 09/716,716 filed Nov. 20, 2000, now abandoned,which is a Continuation of U.S. patent application Ser. No. 09/264,563,now U.S. Pat. No. 6,455,500, which is a Continuation-In-Part of U.S.Provisional Application Nos. 60/077,382 filed Mar. 10, 1998; 60/083670filed Apr. 30, 1998; 60/081,262 filed Apr. 9, 1998; 60/090216 filed Jun.22, 1998; 60/090,096 filed Jun. 19, 1998; 60/096,363 filed Aug. 13,1998; and 60/099,957 filed Sep. 11, 1998.

BACKGROUND OF THE INVENTION

Radiation therapy is currently one of the most useful methods oftreating cancerous tumors. However, radiation therapy damages normaltissue surrounding the tumor (U.S. Pat. No. 5,599,712, incorporated byreference herein in its entirety). This damage can include fibrosis,remodeling of the extracellular matrix, vascular damage, aberrantangiogenesis, pneumonitis, atherogenesis, osteonecrosis, mucositis,immunosuppression and functional impairment (U.S. Pat. No. 5,616,561,incorporated by reference herein in its entirety). As a result of theseradiation-induced side effects, techniques have been developed tominimize radiation-induced damage to surrounding normal tissues bylimiting radiation to the lowest level effective for cancer treatment.Since there is a direct relationship between the amount of radiation andthe effectiveness of the treatment, this method compromises the overalleffectiveness of the treatment.

For some cancer patients, hematopoietic toxicity frequently limits theopportunity for radiation dose escalation (Watanabe et al., British J.Haematol. 94:619-627 (1996)). Repeated or high dose cycles of radiationtherapy may be responsible for severe stem cell depletion leading toimportant long-term hematopoietic sequelea and marrow exhaustion (Masseet al., Blood 91:441-449 (1998). Such stem cell depletion leads todepletion of the full range of hematopoietic lineage specific cells,including megakaryocytes, platelets, monocytes, neutrophils, andlymphocytes, and the resulting complications of such depletion. Forexample, in patients suffering from depressed levels of platelets(thrombocytopenia) the inability to form clots is the most immediate andserious consequence, a potentially fatal complication of many therapiesfor cancer. Such cancer patients are generally treated for this problemwith platelet transfusions. Other patients frequently requiring platelettransfusions are those undergoing bone marrow transplantation orpatients with aplastic anemia. Platelets for such procedures areobtained by plateletpheresis from normal donors. Like most human bloodproducts, platelets for transfusion have a relatively short shelf-lifeand also expose the patients to considerable risk of exposure todangerous viruses, such as the human immunodeficiency virus (HIV).

The administration of hematopoietic growth factors may reduce short-termside effects induced by radiation, but has been hypothesized to causelong-term hematopoietic damage (Masse et al., 1998; Watanabe et al.,1996). Several studies have suggested that co-administration of negativehematopoietic regulators can minimize radiation therapy-inducedmyelotoxicity by reducing the number of progenitor cells that enter thecell cycle. (Watanabe et al., 1996; Dunlop et al., Blood 79:2221-2225(1992); Paukovits et al., Blood 81:1755-1761; Bogden et al., Annals N.Y.Acad. Sci. 628:126-139 (1991); Deeg et al., Ann. Hematol. 74:117-122(1997); Masse et al., 1998). This treatment is based on the premise thathematopoietic stem cells are relatively protected from radiation-relatedtoxicity when quiescent, particularly when the malignant cells areproliferating (Deeg et al., (1997)).

Bone marrow contains pluripotent stem cells that are capable ofreconstituting the entire hematopoietic system. Bone marrowtransplantation has been used to treat various intractable hematopoieticdiseases including leukemia and severe aplastic anemia. (U.S. Pat. No.5,186,931, incorporated by reference herein in its entirety.) Typically,a bone marrow transplant patient is subjected to irradiation to reducethe leukocyte count to zero, followed by transplantation of bone marrowcells which function by producing a sufficient number of normalleukocytes. However, various complications, such as death, infectiousdiseases, graft versus host disease, radiation nephritis, andinterstitial pneumonia frequently occur during the time period betweentransplantation and the return to normal white blood cell levels aftertransplantation.

As a result of these frequent side effects, no satisfactory methods arecurrently available for supporting bone marrow transplantation which arecapable of both increasing survival of bone marrow transplant patientsand also accelerating the reconstitution of the hematopoietic system ofthe patient.

Chronic radiation injuries, such as radiation nephropathy, have beenviewed as inevitable, progressive and untreatable (Moulder et al., BoneMarrow Transplantation 19:729-735 (1997)). The progressive anduntreatable nature of late tissue damage follows from the assumptionthat the injury is due to delayed mitotic cell death resulting fromgenetic injury that is produced and irrevocably fixed in place at thetime of irradiation (Moulder et al., 1997). Under this view, the onlyway to decrease the probability of injury is by limiting the radiationdose or shielding the at risk organs.

However, recent results indicate that late-onset radiation-inducedtissue injury involves complex and dynamic interactions amongparenchymal and vascular cells within a particular organ (Moulder etal., 1997). This model of chronic radiation injury suggests thatpharmacological intervention after radiation exposure would beeffective.

Thus, despite advances in the field of radiation therapy, prior artmethods have proven to be of limited utility in minimizingradiation-induced tissue damage, and improving the efficacy of tumorradiation therapy and bone marrow transplantation. Thus, there is a needfor improved therapeutic methods to mitigate radiation induced tissuedamage and to improve the effectiveness of radiation therapy.Furthermore, the ability to stimulate endogenous platelet formation inthrombocytopenic patients with a concomitant reduction in theirdependence on platelet transfusion would be of great benefit. Inaddition the ability to correct or prevent thrombocytopenia in patientsundergoing radiation therapy or chemotherapy for cancer would make suchtreatments safer and possibly permit increases in the intensity of thetherapy thereby yielding greater anti-cancer effects.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods and kits formitigating radiation induced tissue damage, improving the effectivenessof radiation therapy, to support bone marrow transplantation, andpromoting megakaryocyte production and mobilization and plateletproduction, each method comprising the administration ofangiotensinogen, angiotensin I (AI), AI analogues, AI fragments andanalogues thereof, angiotensin II (AII), AII analogues, AII fragments oranalogues thereof or AII AT₂ type 2 receptor agonists to a patient inneed thereof.

In another aspect of the present invention, an improved cell culturemedium and kits are provided for the production of megakaryocytes andplatelets wherein the improvement comprises addition to the cell culturemedium of an effective amount of angiotensinogen, AI, AI analogues, AIfragments and analogues thereof, AII, AII analogues, AII fragments oranalogues thereof or AII AT₂ type 2 receptor agonists.

These aspects and other aspects of the invention become apparent inlight of the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the effect of AII treatment two days prior toexposure on post-irradiation mouse mortality.

FIG. 2 is a graph showing the effect of AII treatment on the day ofexposure on post-irradiation mouse mortality.

FIG. 3 is a graph showing the effect of AII treatment two days followingexposure on post-irradiation mouse mortality.

FIG. 4 is a graph showing the effect of AII treatment two days prior toexposure on white blood cell number after irradiation.

FIG. 5 is a graph showing the effect of AII treatment on the day ofexposure on white blood cell number after irradiation.

FIG. 6 is a graph showing the effect of AII treatment two days followingexposure on white blood cell number after irradiation.

FIG. 7 is a graph showing the effect of AII treatment two days prior toexposure on megakaryocyte number after irradiation.

FIG. 8 is a graph showing the effect of AII treatment on the day ofexposure on megakaryocyte number after irradiation.

FIG. 9 is a graph showing the effect of AII treatment two days followingexposure on megakaryocyte percentage after irradiation.

FIG. 10 is a graph showing the effect of AII treatment two days prior toexposure on monocyte number after irradiation.

FIG. 11 is a graph showing the effect of AII treatment on the day ofexposure on monocyte number after irradiation.

FIG. 12 is a graph showing the effect of AII treatment two daysfollowing exposure on monocyte number after irradiation.

FIG. 13 is a graph showing the effect of AII treatment two days prior toexposure on neutrophil number after irradiation.

FIG. 14 is a graph showing the effect of AII treatment on the day ofexposure on neutrophil number after irradiation.

FIG. 15 is a graph showing the effect of AII treatment two daysfollowing exposure on neutrophil number after irradiation.

FIG. 16 is a graph showing the effect of AII treatment two days prior toexposure on lymphocyte number after irradiation.

FIG. 17 is a graph showing the effect of AII treatment on the day ofexposure on lymphocyte number after irradiation.

FIG. 18 is a graph showing the effect of AII treatment two daysfollowing exposure on lymphocyte number after irradiation.

FIG. 19 is a graph showing is a graph showing the effect of AIIanalogues and fragments treatment on white blood cell number afterirradiation.

FIG. 20 is a graph showing is a graph showing the effect of AIIanalogues and fragments treatment on platelet number after irradiation.

FIG. 21 is a graph showing the effect of AII on mouse survival receivingbone marrow transplantation after lethal irradiation.

FIG. 22 is a graph showing the effect of AII analogues and fragmentstreatment on white blood cell number after irradiation.

FIG. 23 is a graph showing the effect of AII on white blood cell numberin the blood of mice receiving bone marrow transplantation after lethalirradiation.

FIG. 24 is a graph showing the effect of AII on white blood cell numberin the blood of mice receiving bone marrow transplantation after lethalirradiation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

AII references patents and patent applications are hereby incorporatedby reference in their entirety.

The present invention fulfills the needs for improved therapeuticmethods to mitigate radiation induced tissue damage, to improve theeffectiveness of radiation therapy, to support bone marrowtransplantation, and to promote megakaryocyte production andmobilization and platelet production.

As defined herein the phrase “mitigation of tissue damage” refers notonly to reduction of damage, but also encompasses recovery of tissuefrom damage. As used herein “tissue” refers to any tissue type, and alsoincludes hematopoietic stem and progenitor cells, white blood cells andplatelets.

As defined herein the term “megakaryocyte mobilization” refers to themovement of a megakaryocyte precursor cell from the bone marrow into theperiphery.

As defined herein, the phrase “improved platelet production” or“improved megakaryocyte production,” means that the number of plateletsor megakaryocytes is significantly elevated above the normal range ofplatelets or megakaryocytes in the particular mammal involved. Theelevation of platelet or megakaryocyte counts may occur in atime-dependent manner, and may be cyclical, increasing and then constantor decreasing, or constant, etc.

Unless otherwise indicated, the term “active agents” as used hereinrefers to the group of compounds comprising angiotensinogen, angiotensinI (AI), AI analogues, AI fragments and analogues thereof, angiotensin II(AII), AII analogues, AII fragments or analogues thereof and AII AT₂type 2 receptor agonists.

Within this application, unless otherwise stated, the techniquesutilized may be found in any of several well-known references such as:Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, ColdSpring Harbor Laboratory Press), Gene Expression Technology (Methods inEnzymology, Vol. 185, edited by D. Goeddel, 1991, Academic Press, SanDiego, Calif.), “Guide to Protein Purification” in Methods in Enzymology(M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: AGuide to Methods and Applications (Innis, et al. 1990. Academic Press,San Diego, Calif.), Culture of Animal Cells. A Manual of BasicTechnique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.),Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray,The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog(Ambion, Austin, Tex.).

U.S. Pat. No. 5,015,629 to DiZerega (the entire disclosure of which ishereby incorporated by reference) describes a method for increasing therate of healing of wound tissue, comprising the application to suchtissue of angiotensin II (AII) in an amount which is sufficient for saidincrease. The application of AII to wound tissue significantly increasesthe rate of wound healing, leading to a more rapid re-epithelializationand tissue repair. The term AII refers to an octapeptide present inhumans and other species having the sequenceAsp-Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:1]. The biological formationof angiotensin is initiated by the action of renin on the plasmasubstrate angiotensinogen. The substance so formed is a decapeptidecalled angiotensin I (AI) which is converted to AII by the convertingenzyme angiotensinase which removes the C-terminal His-Leu residues fromAI (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu [SEQ ID NO:37]). All is aknown pressor agent and is commercially available. The use of AIIanalogues and fragments, AT2 agonists, as well as AIII and AIIIanalogues and fragments in wound healing has also been described. (U.S.Pat. No. 5,629,292; U.S. Pat. No. 5,716,935; WO 96/39164; all referencesherein incorporated by reference in their entirety.)

Studies have shown that AII increases mitogenesis and chemotaxis incultured cells that are involved in wound repair, and also increasestheir release of growth factors and extracellular matrices (dizerega,U.S. Pat. No. 5,015,629; Dzau et. al., J. Mol. Cell. Cardiol. 21:S7(Supp III) 1989; Berk et. al., Hypertension 13:305-14 (1989); Kawahara,et al., BBRC 150:52-9 (1988); Naftilan, et al., J. Clin. Invest.83:1419-23 (1989); Taubman et al., J. Biol. Chem 264:526-530 (1989);Nakahara, et al., BBRC 184:811-8 (1992); Stouffer and Owens, Circ. Res.70:820 (1992); Wolf, et al., Am. J. Pathol. 140:95-107 (1992); Bell andMadri, Am. J. Pathol. 137:7-12 (1990). In addition, AII was shown to beangiogenic in rabbit corneal eye and chick chorioallantoic membranemodels (Fernandez, et al., J. Lab. Clin. Med. 105:141 (1985); LeNoble,et al., Eur. J. Pharmacol. 195:305-6 (1991). Therefore, AII mayaccelerate wound repair through increased neovascularization, growthfactor release, reepithelialization and/or production of extracellularmatrix.

AII has also been implicated in both cell growth and differentiation(Meffert et al., Mol. and Cellul. Endocrin. 122:59 (1996)). Two mainclasses of AII receptors, AT₁ and AT₂ have been identified (Meffert,1996). The growth-promoting effects of AII have been attributed tomediation by the AT1 receptor, while some evidence suggests that the AT2receptor may be involved in mediation of the cell differentiationeffects of AII (Bedecs et al., Biochem. J. 325:449 (1997)).

The effects of AII receptor and AII receptor antagonists have beenexamined in two experimental models of vascular injury and repair whichsuggest that both AII receptor subtypes (AT1 and AT2) play a role inwound healing (Janiak et al., Hypertension 20:737-45 (1992); Prescott,et al., Am. J. Pathol. 139:1291-1296 (1991); Kauffman, et al., Life Sci.49:223-228 (1991); Viswanathan, et al., Peptides 13:783-786 (1992);Kimura, et al., BBRC 187:1083-1090 (1992).

Many studies have focused upon AII(1-7) (AII residues 1-7) or otherfragments of AII to evaluate their activity. AII(1-7) elicits some, butnot the full range of effects elicited by AII. Pfeilschifter, et al.,Eur. J. Pharmacol. 225:57-62 (1992); Jaiswal, et al., Hypertension19(Supp. II):II-49-II-55 (1992); Edwards and Stack, J. Pharmacol. Exper.Ther. 266:506-510 (1993); Jaiswal, et al., J. Pharmacol. Exper. Ther.265:664-673 (1991); Jaiswal, et al., Hypertension 17:1115-1120 (1991);Portsi, et al., Br. J. Pharmacol. 111:652-654 (1994).

While a single pilot study has suggested that AII-induced hypertensionmight be effective in combination with radiation therapy in thetreatment of lung cancer patients (Kato et al., Radiation Medicine11:86-90 (1993)), many studies have demonstrated that antagonists ofangiotensin converting enzyme (ACE), which mediate the production ofAII, are effective in reducing radiation nephropathy, bone marrowtransplantation nephropathy, and acute radiation injury (Moulder et al.,Int. J. Radiation One. Biol. Phys. 27:93-99 (1993); Moulder et al., BoneMarrow Transpl. 19:729-735 (1997); Moulder et al., Radiation Res.146:106-110 (1996); Cohen et al., J. Lab. Clin. Med. 129:536-547 (1997);Moulder et al., Radiation Res. 136:404-407 (1993); Yoon et al., Int. J.Radiat. Oncol. Biol. Phys. 30:873-878 (1994); Ward et al., RadiationRes. 135:81-87 (1993); Cohen et al., Lab. Invest. 75:349-360 (1996);Cohen et al., J. Lab. Clin. Med. 124:371-380 (1994); Gerarci et al.,Radiation Res. 143:58-68 (1995)). The effect of the ACE inhibitors hasbeen demonstrated, in at least one case, to be directly caused by thereduction of activation of the AT1 receptor by AII (Moulder et al.,Radiation Res. 146:106-110 (1996)). These results have led to thesuggestion that, in the case of radiation nephropathy, the mosteffective treatment is the use of ACE inhibitors (Moulder et al., BoneMarrow Transplantation 19:729-735 (1997)).

Furthermore, it has recently been demonstrated that angiotensinogen,angiotensin I (AI), AI analogues, AI fragments and analogues thereof,AII, AII analogues, AII fragments or analogues thereof or AII AT₂ type 2receptor agonists are potent stimulators of hematopoietic stem cellproliferation (U.S. patent application Ser. No. 09/012,400, herebyincorporated by reference in its entirety). Therefore, it would beexpected that the use of these compounds might cause long-termhematopoietic damage if used in conjunction with radiation therapy(Masse et al., 1998; Watanabe et al., 1996).

Based on all of the above, it would be unexpected that the use ofangiotensinogen, angiotensin I (AI), AI analogues, AI fragments andanalogues thereof, AII, AII analogues, AII fragments or analoguesthereof or AII AT₂ type 2 receptor agonists would be effective inreducing radiation-induced human tissue damage or in treating patientsin need of radiation therapy.

None of these studies teach or suggest the use of angiotensinogen,angiotensin I (AI), AI analogues, AI fragments and analogues thereof,angiotensin II (AII), AII analogues, AII fragments or analogues thereofor AII AT₂ type 2 receptor agonists to stimulate the production andmobilization of megakaryocytes, or to stimulate the production ofplatelets.

A peptide agonist selective for the AT2 receptor (AII has 100 timeshigher affinity for AT2 than AT1) is p-aminophenylalanine6-AII[“(p-NH₂-Phe)6-AII)”], Asp-Arg-Val-Tyr-Ile-Xaa-Pro-Phe [SEQ ID NO.36]wherein Xaa is p-NH₂-Phe (Speth and Kim, BBRC 169:997-1006 (1990). Thispeptide gave binding characteristics comparable to AT2 antagonists inthe experimental models tested (Catalioto, et al., Eur. J. Pharmacol.256:93-97 (1994); Bryson, et al., Eur. J. Pharmacol. 225:119-127 (1992).

The active AI, AI analogues, AI fragments and analogues thereof, AIIanalogues, fragments of AII and analogues thereof of particular interestin accordance with the present invention are characterized as comprisinga sequence consisting of at least three contiguous amino acids of groupsR¹-R⁸ in the sequence of general formula IR¹—R²—R³—R⁴—R⁵—R⁶—R⁷—R⁸

-   -   in which R¹ and R² together form a group of formula        X—R^(A)—R^(B)—,    -   wherein X is H or a one to three peptide group,    -   R^(A) is suitably selected from Asp, Glu, Asn, Acpc        (1-aminocyclopentane carboxylic acid), Ala, Me²Gly, Pro, Bet,        Glu(NH₂), Gly, Asp(NH₂) and Suc,    -   R^(B) is suitably selected from Arg, Lys, Ala, Om, Ser(Ac), Sar,        D-Arg and D-Lys;    -   R³ is selected from the group consisting of Val, Ala, Leu,        norLeu, Ile, Gly, Pro, Aib, Acpc, Lys, and Tyr;    -   R⁴ is selected from the group consisting of Tyr, Tyr(PO₃)₂, Thr,        Ser, homoSer, Ala, and azaTyr;    -   R⁵ is selected from the group consisting of Ile, Ala, Leu,        norLeu, Val and Gly;    -   R⁶ is His, Arg or 6-NH₂-Phe;    -   R⁷ is Pro or Ala; and    -   R⁸ is selected from the group consisting of Phe, Phe(Br), Ile        and Tyr, excluding sequences including R⁴ as a terminal Tyr        group.

Compounds falling within the category of AT2 agonists useful in thepractice of the invention include the AII analogues set forth abovesubject to the restriction that R⁶ is p-NH₂-Phe. In addition to peptideagents, various nonpeptidic agents (e.g., peptidomimetics) having therequisite AT2 agonist activity are further contemplated for use inaccordance with the present invention.

Particularly preferred combinations for R^(A) and R^(B) are Asp-Arg,Asp-Lys, Glu-Arg and Glu-Lys. Particularly preferred embodiments of thisclass include the following: AII, AIII or AII(2-8),Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2]; AII(3-8), also known asdesl-AIII or AIV, Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:3]; AII(1-7),Asp-Arg-Val-Tyr-Ile-His-Pro [SEQ ID NO:4]; AII(2-7).Arg-Val-Tyr-Ile-His-Pro [SEQ ID NO:5]; AII(3-7), Val-Tyr-Ile-His-Pro[SEQ ID NO:6]; AII(5-8), Ile-His-Pro-Phe [SEQ ID NO:7]; AII(1-6),Asp-Arg-Val-Tyr-Ile-His [SEQ ID NO:8]; AII(1-5), Asp-Arg-Val-Tyr-Ile[SEQ ID NO:9]; AII(1-4), Asp-Arg-Val-Tyr [SEQ ID NO:10]; and AII(1-3),Asp-Arg-Val [SEQ ID NO:11]. Other preferred embodiments include:Arg-norLeu-Tyr-Ile-His-Pro-Phe [SEQ ID NO:12] andArg-Val-Tyr-norLeu-His-Pro-Phe [SEQ ID NO:13]. Still another preferredembodiment encompassed within the scope of the invention is a peptidehaving the sequence Asp-Arg-Pro-Tyr-Ile-His-Pro-Phe [SEQ ID NO:31].AII(6-8), His-Pro-Phe [SEQ ID NO:14] and AII(4-8), Tyr-Ile-His-Pro-Phe[SEQ ID NO:15] were also tested and found not to be effective.

A class of particularly preferred compounds in accordance with thepresent invention consists of those with the following generalstructure:R1-Arg-R2-R3-R4-His-Pro-R5

wherein R1 is selected from the group consisting of H and Asp;

R2 is selected from the group consisting of Val and Pro;

R3 is selected from the group consisting of Tyr and Tyr(PO₃)₂;

R4 is selected from the group consisting of Ala, Ile, Leu, and norLeu;and

R5 is Phe, Ile, or is absent.

Particularly preferred embodiment of this class are selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:18, SEQ IDNO:26, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:38

Another class of compounds of particular interest in accordance with thepresent invention are those of the general formula IIR²—R³—R⁴—R⁵—R⁶—R⁷—R⁸

-   -   in which R² is selected from the group consisting of H, Arg,        Lys, Ala, Om, Ser(Ac), Sar, D-Arg and D-Lys;    -   R³ is selected from the group consisting of Val, Ala, Leu,        norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr;    -   R⁴ is selected from the group consisting of Tyr, Tyr(PO₃)₂, Thr,        Ser, homoSer and azaTyr;    -   R⁵ is selected from the group consisting of Ile, Ala, Leu,        norLeu, Val and Gly;    -   R⁶ is His, Arg or 6-NH₂-Phe;    -   R⁷ is Pro or Ala; and    -   R⁸ is selected from the group consisting of Phe, Phe(Br), Ile        and Tyr.

A particularly preferred subclass of the compounds of general formula IIhas the formula

R²-R³-Tyr-R⁵-His-Pro-Phe [SEQ ID NO: 16]

wherein R², R³ and R⁵ are as previously defined. Particularly preferredis angiotensin III of the formula Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ IDNO:2]. Other preferred compounds include peptides having the structuresArg-Val-Tyr-Gly-His-Pro-Phe [SEQ ID NO:17] andArg-Val-Tyr-Ala-His-Pro-Phe [SEQ ID NO:18]. The fragment AII(4-8) wasineffective in repeated tests; this is believed to be due to the exposedtyrosine on the N-terminus.

In the above formulas, the standard three-letter abbreviations for aminoacid residues are employed. In the absence of an indication to thecontrary, the L-form of the amino acid is intended. Other residues areabbreviated as follows:

TABLE 1 Abbreviation for Amino Acids Me²Gly N,N-dimethylglycyl Bet1-carboxy-N,N,N-trimethylmethanaminium hydroxide inner salt (betaine)Sue Succinyl Phe(Br) p-bromo-L-phenylalanyl azaTyr aza-α′-homo-L-tyrosylAcpc 1-aminocyclopentane carboxylic acid Aib 2-aminoisobutyric acid SarN-methylglycyl (sarcosine)

It has been suggested that AII and its analogues adopt either a gamma ora beta turn (Regoli, et al., Pharmacological Reviews 26:69 (1974). Ingeneral, it is believed that neutral side chains in position R³, R⁵ andR⁷ may be involved in maintaining the appropriate distance betweenactive groups in positions R⁴, R⁶ and R⁸ primarily responsible forbinding to receptors and/or intrinsic activity. Hydrophobic side chainsin positions R³, R⁵ and R⁸ may also play an important role in the wholeconformation of the peptide and/or contribute to the formation of ahypothetical hydrophobic pocket.

Appropriate side chains on the amino acid in position R² may contributeto affinity of the compounds for target receptors and/or play animportant role in the conformation of the peptide. For this reason, Argand Lys are particularly preferred as R².

For purposes of the present invention, it is believed that R³ may beinvolved in the formation of linear or nonlinear hydrogen bonds with R⁵(in the gamma turn model) or R⁶ (in the beta turn model). R³ would alsoparticipate in the first turn in a beta antiparallel structure (whichhas also been proposed as a possible structure). In contrast to otherpositions in general formula I, it appears that beta and gamma branchingare equally effective in this position. Moreover, a single hydrogen bondmay be sufficient to maintain a relatively stable conformation.Accordingly, R³ may suitably be selected from Val, Ala, Leu, norLeu,Ile, Gly, Pro, Aib, Acpc and Tyr. In another preferred embodiment, R³ isLys.

With respect to R⁴, conformational analyses have suggested that the sidechain in this position (as well as in R³ and R⁵) contribute to ahydrophobic cluster believed to be essential for occupation andstimulation of receptors. Thus, R⁴ is preferably selected from Tyr, Thr,Tyr (PO₃)₂, homoSer, Ser and azaTyr. In this position, Tyr isparticularly preferred as it may form a hydrogen bond with the receptorsite capable of accepting a hydrogen from the phenolic hydroxyl (Regoli,et al. (1974), supra). In a further preferred embodiment, R⁴ is Ala.

In position R⁵, an amino acid with a β aliphatic or alicyclic chain isparticularly desirable. Therefore, while Gly is suitable in position R⁵,it is preferred that the amino acid in this position be selected fromIle, Ala, Leu, norLeu, Gly and Val.

In the AI, AI analogues, AI fragments and analogues thereof, AII, AIIanalogues, fragments and analogues of fragments of particular interestin accordance with the present invention, R⁶ is His, Arg or 6-NH₂-Phe.The unique properties of the imidazole ring of histidine (e.g.,ionization at physiological pH, ability to act as proton donor oracceptor, aromatic character) are believed to contribute to itsparticular utility as R⁶. For example, conformational models suggestthat His may participate in hydrogen bond formation (in the beta model)or in the second turn of the antiparallel structure by influencing theorientation of R⁷. Similarly, it is presently considered that R⁷ shouldbe Pro in order to provide the most desirable orientation of R⁸. Inposition R⁸, both a hydrophobic ring and an anionic carboxyl terminalappear to be particularly useful in binding of the analogues of interestto receptors; therefore, Tyr and especially Phe are preferred forpurposes of the present invention.

Analogues of particular interest include the following:

TABLE 2 Angiotensin II Analogues All Analogue Sequence Name Amino AcidSequence Identifier Analogue 1 Asp-Arg-Val-Tyr-Val-His- SEQ ID NO: 19Pro-Phe Analogue 2 Asn-Arg-Val-Tyr-Val-His- SEQ ID NO: 20 Pro-PheAnalogue 3 Ala-Pro-Gly-Asp-Arg-Ile- SEQ ID NO: 21 Tyr-Val-His-Pro-PheAnalogue 4 Glu-Arg-Val-Tyr-Ile-His- SEQ ID NO: 22 Pro-Phe Analogue 5Asp-Lys-Val-Tyr-Ile-His- SEQ ID NO: 23 Pro-Phe Analogue 6Asp-Arg-Ala-Tyr-Ile-His- SEQ ID NO: 24 Pro-Phe Analogue 7Asp-Arg-Val-Thr-Ile-His- SEQ ID NO: 25 Pro-Phe Analogue 8Asp-Arg-Val-Tyr-Leu-His- SEQ ID NO: 26 Pro-Phe Analogue 9Asp-Arg-Val-Tyr-Ile-Arg- SEQ ID NO: 27 Pro-Phe Analogue 10Asp-Arg-Val-Tyr-Ile-His- SEQ ID NO: 28 Ala-Phe Analogue 11Asp-Arg-Val-Tyr-Ile-His- SEQ ID NO: 29 Pro-Tyr Analogue 12Pro-Arg-Val-Tyr-Ile-His- SEQ ID NO: 30 Pro-Phe Analogue 13Asp-Arg-Pro-Tyr-Ile-His- SEQ ID NO: 31 Pro-Phe Analogue 14Asp-Arg-Val-Tyr(PO₃)₂-Ile- SEQ ID NO: 32 His-Pro-Phe Analogue 15Asp-Arg-norLeu-Tyr-Ile- SEQ ID NO: 33 His-Pro-Phe Analogue 16Asp-Arg-Val-Tyr-norLeu- SEQ ID NO: 34 His-Pro-Phe Analogue 17Asp-Arg-Val-homoSer-Tyr- SEQ ID NO: 35 Ile-His-Pro-Phe

The polypeptides of the instant invention may be synthesized by methodssuch as those set forth in J. M. Stewart and J. D. Young, Solid PhasePeptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, Ill. (1984)and J. Meienhofer, Hormonal Proteins and Peptides, Vol. 2, AcademicPress, New York, (1973) for solid phase synthesis and E. Schroder and K.Lubke, The Peptides, Vol. 1, Academic Press, New York, (1965) forsolution synthesis. The disclosures of the foregoing treatises areincorporated by reference herein.

In general, these methods involve the sequential addition of protectedamino acids to a growing peptide chain (U.S. Pat. No. 5,693,616, hereinincorporated by reference in its entirety). Normally, either the aminoor carboxyl group of the first amino acid and any reactive side chaingroup are protected. This protected amino acid is then either attachedto an inert solid support, or utilized in solution, and the next aminoacid in the sequence, also suitably protected, is added under conditionsamenable to formation of the amide linkage. After all the desired aminoacids have been linked in the proper sequence, protecting groups and anysolid support are removed to afford the crude polypeptide. Thepolypeptide is desalted and purified, preferably chromatographically, toyield the final product.

In one aspect, the present invention provides methods and kits for themitigation of tissue damage due to radiation exposure comprising theadministration of angiotensinogen, angiotensin I (AI), AI analogues, AIfragments and analogues thereof, angiotensin II (AII), AII analogues,AII fragments or analogues thereof or AII AT₂ type 2 receptor agonists(the “active agents”).

In another aspect, the present invention provides improved methods andkits for treating a patient afflicted with a neoplastic disease statethat is being treated with ionizing or nonionizing radiation, theimprovement comprising conjunctive therapy whereby an effectiveradioprotective amount of the active agents is provided.

In another aspect, the present invention provides improved methods andkits for treating a patient in need of radiation therapy, theimprovement comprising the administration of the active agents inconjunction with the radiation therapy.

The invention is appropriate for use with any type of ionizing radiationexposure such as therapeutic or accidental X-ray, gamma ray, or betaparticle exposure. Examples of ionizing radiation exposure suitable fortreatment with the methods and kits of the present invention include,but are not limited to, clinical radiation therapy, medical diagnosticsusing radioactive tracers, exposure to naturally occurring ionizingradiation sources such as uranium and radon, wartime exposure, andaccidental exposures including occupational exposure at nuclear powerfacilities, and medical and research institutions. Examples ofnonionizing radiation exposure suitable for treatment with the methodsand kits of the present invention include, but are not limited to,ultraviolet light, X-rays, microwaves, radio-frequency waves, andelectromagnetic radiation.

Virtually any tissue susceptible to radiation-induced tissue damage cangain protection by use of the active agents of the invention. Forexample, breast tissue is an excellent candidate for receiving thebenefit of the subject invention. Radiation-induced tissue damage can bea fatal side effect of over-exposure to radiation therapy. Typically,the fibrotic reaction common in normal breast tissue surrounding thecancerous tumor being treated with radiation therapy undermines thecosmetic advantages of radiation therapy over surgical treatment. Thisdisadvantage will lead many patients to elect a less effective or moredangerous treatment after radiation therapy.

The present invention is also particularly suitable for those patientsin need of repeated or high doses of radiation therapy. For some cancerpatients, hematopoietic toxicity frequently limits the opportunity forradiation dose escalation (Watanabe et al., British J. Haematol.94:619-627 (1996)). Repeated or high dose cycles of radiation therapymay be responsible for severe stem cell depletion leading to importantlong-term hematopoietic sequelea and marrow exhaustion. The methods ofthe present invention provide for improved mortality and blood cellcount when used in conjunction with radiation therapy.

Skin exposure is particularly common in accidental radiation exposure.It is an excellent candidate for the inventive therapy, especially asthe compounds of the invention can be administered topically. Othertissues that are susceptible to radiation-induced damage followingaccidental or therapeutic ionizing or nonionizing radiation exposureinclude, but are not limited to: liver, lung, gastrointestinal tract,kidneys, testes, salivary gland, mucosa and brain.

In another aspect, the present invention provides improved methods andkits for supporting bone marrow transplantation comprising theadministration of the active agents to a patient in need thereof. Thesecompounds may be administered in combination with auxiliary agentsincluding, but not limited to interleukin (IL)-3, IL-1, IL-4, Il-5,granulocyte colony stimulating factor (G-CSF), granulocyte-macrophagecolony stimulating factor (GM-CSF), macrophage colony stimulating factor(M-CSF), anticancer agents, antiviral agents, and antibiotics.

In a further aspect, the present invention provides kits for mitigatingradiation induced tissue damage and improving the efficacy of radiationtherapy, wherein the kits comprise an effective amount of the activeagents of the invention for mitigating radiation induced tissue damageor improving the efficacy of radiation therapy, and instructions forusing the amount effective of active agent as a therapeutic. In apreferred embodiment, the kit further comprises a pharmaceuticallyacceptable carrier, such as those adjuvants described above. In anotherpreferred embodiment, the kit further comprises a means for delivery ofthe active agent to a patient. Such devices include, but are not limitedto syringes, matrical or micellar solutions, bandages, wound dressings,aerosol sprays, lipid foams, transdermal patches, topical administrativeagents, polyethylene glycol polymers, carboxymethyl cellulosepreparations, crystalloid preparations (e.g., saline, Ringer's lactatesolution, phosphate-buffered saline, etc.), viscoelastics, polyethyleneglycols, and polypropylene glycols. The means for delivery may eithercontain the effective amount of angiotensinogen, AI, AI analogues, AIfragments and analogues thereof, AII, AII analogues, AII fragments oranalogues thereof or AII AT₂ type 2 receptor agonists, or may beseparate from the compounds, which are then applied to the means fordelivery at the time of use.

The methods and kits of the present invention, by mitigating radiationinduced tissue damage and improving the efficacy of radiation therapyand bone marrow transplantation, significantly enhance the utility ofpresently available treatments both for radiation-induced tissue damageand for clinical radiation therapy.

In a further aspect of the present invention, a method of increasingmegakaryocyte production and mobilization and platelet production byexposure to the active agents of the inventions is disclosed. In oneembodiment, megakaryocytes are isolated from bone marrow as described inU.S. Pat. No. 5,178,856, incorporated by reference herein in itsentirety. Briefly, marrow is flushed from a subject's femur withIscove's modification of Dulbecco's medium (IMDM) supplemented withNutridoma-SP (Boehringer Mannheim, Indianapolis, Ind.), a serum-freemedium supplement. For culture studies, a single cell suspension is madeby repetitive expulsion through progressively smaller needles. For flowcytometry controls, a monocellular suspension is made by gentlefiltration through a 100 micron nylon mesh. Preferably, adherent cellsare removed to enrich the numbers of megakaryocytes or their progenitorcells. Up to 2×10⁶ cells/ml are placed in growth medium at 37° C. in ahumidified atmosphere in the presence of, preferably, between about 0.1ng/ml and about 10 mg/ml of the active agents. The cells are expandedfor a period of between 2 and 21 days and cellular proliferation isassessed at various time points during this time period. Subsequentmedium changes are performed as needed. In a preferred embodiment,megakaryocyte production and mobilization and platelet production areassessed by the extent of megakaryocyte ploidization by flow cytometryas described in U.S. Pat. No. 5,155,211, incorporated by referenceherein in its entirety. Briefly, the appearance of granules and theextensive surface-connected open canalicular membrane system as well asa substantial decrease in the nucleus:cytoplasm volume distribution,indicates that the megakaryocyte population has completed the process ofpolyploidization but has not yet generated a major portion of theirfinal complement of platelet-specific cytoplasmic components.

In another embodiment, subjects are irradiated as above and active agentis injected subcutaneously before, at the time of, and afterirradiation. Blood samples are taken at various times afteradministration of the active agent to monitor the number of white bloodcells, megakaryocytes and platelets. In a preferred embodiment, subjectsare treated with total body irradiation and active agent is administeredsubcutaneously (10 μg/kg/day or 100 μg/kg/day) at various times beforeand after irradiation. The number of white blood cells, megakaryocytesand platelets is preferably determined by counting with a hemacytometerfollowed by differential morphologic analysis.

In another embodiment of this aspect of the invention, hematopoieticprecursor cells are isolated from bone marrow, peripheral blood orumbilical cord blood and cultured under appropriate growth conditions,in the presence of the active agents. Megakaryocyte production isassessed at various time points during culture by differentialmorphologic analysis.

In a preferred embodiment, hematopoietic precursor cells are isolatedfrom bone marrow aspirates from the posterior iliac crest (Caplan andHaynesworth, U.S. Pat. No. 5,486,359). CD34⁺ hematopoietic precursorcells are isolated from the aspirate by attaching a biotinylatedmonoclonal antibody specific for CD34 (available from Becton Dickinson,Sunnyvale, Calif., USA) to a streptavidin affinity column (Ceprate SC;CellPro, Bothell, WA, USA) and passing the aspirate through the column,followed by appropriate column washing and stripping, according tostandard techniques in the art. The isolated cells are suspended inculture medium and incubated in the presence of, preferably, betweenabout 0.1 ng/ml and about 10 mg/ml of the active agents of theinvention. The cells are expanded for a period of between 8 and 21 daysand megakaryocyte production is assessed via phase microscopy to detectincreased size and polyploidization at various points during this timeperiod.

In a further embodiment of the present invention, a method of increasingmegakaryocyte production and mobilization and platelet production byexposure to the active agents is disclosed, either in the presence orabsence of other growth factors and cytokines. Examples of such growthfactors and cytokines include, but are not limited to thrombopoietin,lymphokines, interleukins—1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, granulocytecolony-stimulating factor, granulocyte/macrophage colony stimulatingfactor, macrophage colony-stimulating factor, tumor necrosis factor,epidermal growth factor, fibroblast growth factor, platelet derivedgrowth factor, transforming growth factor beta, and stem cell factor.

In a further preferred embodiment, megakaryocytes and/or platelets thathave been cultured in the presence of the active agents are used forautologous transplantation, to reconstitute a depleted hematopoieticsystem. Prior to transplantation, the cells are rinsed to remove alltraces of culture fluid, resuspended in an appropriate medium and thenpelleted and rinsed several times. After the final rinse, the cells areresuspended at between 0.7×10⁶ and 50×10⁶ cells per ml in an appropriatemedium and reinfused into a subject through intravenous infusions.Following transplantation, subject peripheral blood samples areevaluated for increased megakaryocyte ploidy and platelet number bystandard flow cytometry and cell sorting techniques. (Talmadge, et al.,supra).

In another aspect of the present invention the active agents are used toincrease in vivo megakaryocyte production and mobilization and plateletproduction. For use in increasing megakaryocyte production andmobilization and platelet production, the active agents may beadministered by any suitable route, including orally, parentally, byinhalation spray, rectally, or topically in dosage unit formulationscontaining conventional pharmaceutically acceptable carriers, adjuvants,and vehicles. The term parenteral as used herein includes, subcutaneous,intravenous, intramuscular, intrasternal, intratendinous, intraspinal,intracranial, intrathoracic, infusion techniques or intraperitoneally.

The active agents of all aspects of the present invention may beadministered by any suitable route, including orally, parentally, byinhalation spray, rectally, or topically in dosage unit formulationscontaining conventional pharmaceutically acceptable carriers, adjuvants,and vehicles. The term parenteral as used herein includes, subcutaneous,intravenous, intraarterial, intramuscular, intrasternal, intratendinous,intraspinal, intracranial, intrathoracic, infusion techniques orintraperitoneally.

The active agents of the invention may be made up in a solid form(including granules, powders or suppositories) or in a liquid form(e.g., solutions, suspensions, or emulsions). The compounds of theinvention may be applied in a variety of solutions. Suitable solutionsfor use in accordance with the invention are sterile, dissolvesufficient amounts of the peptide, and are not harmful for the proposedapplication. In this regard, the compounds of the present invention arevery stable but are hydrolyzed by strong acids and bases. The compoundsof the present invention are soluble in organic solvents and in aqueoussolutions at pH 5-8.

The active agents may be subjected to conventional pharmaceuticaloperations such as sterilization and/or may contain conventionaladjuvants, such as preservatives, stabilizers, wetting agents,emulsifiers, buffers etc. For administration, the active agents areordinarily combined with one or more adjuvants appropriate for theindicated route of administration. The compounds may be admixed withlactose, sucrose, starch powder, cellulose esters of alkanoic acids,stearic acid, talc, magnesium stearate, magnesium oxide, sodium andcalcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodiumalginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, and tabletedor encapsulated for conventional administration. Alternatively, thecompounds of this invention may be dissolved in saline, water,polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidalsolutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil,tragacanth gum, and/or various buffers. Other adjuvants and modes ofadministration are well known in the pharmaceutical art. The carrier ordiluent may include time delay material, such as glyceryl monostearateor glyceryl distearate alone or with a wax, or other materials wellknown in the art.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin(e.g., liniments, lotions, ointments, creams, or pastes) and dropssuitable for administration to the eye, ear, or nose.

The dosage regimen for mitigating radiation-induced tissue damage andimproving the efficacy of radiation therapy with the active agents isbased on a variety of factors, including the type of injury, the age,weight, sex, medical condition of the individual, the severity of thecondition, the route of administration, and the particular compoundemployed. Thus, the dosage regimen may vary widely, but can bedetermined routinely by a physician using standard methods. Dosagelevels of the order of between 0.1 ng/kg and 10 mg/kg body weight of theactive agents are useful for all methods of use disclosed herein.

The treatment regime will also vary depending on the disease beingtreated, based on a variety of factors, including the type of injury,the age, weight, sex, medical condition of the individual, the severityof the condition, the route of administration, and the particularcompound employed. For example, the active agents are administered to anoncology patient for up to 30 days prior to a course of radiationtherapy and for up to 60 days post-radiation exposure. The therapy isadministered for 1 to 6 times per day at dosages as described above.

In all of these embodiments, the compounds of the invention can beadministered either prior to, simultaneously with, or subsequent toradiation exposure.

In a preferred embodiment, the active agent is administeredsubcutaneously. A suitable subcutaneous dose of active ingredient ofactive agent is preferably between about 0.1 ng/kg and about 10 mg/kgadministered twice daily for a time sufficient to mitigateradiation-induced tissue damage, to provide a radioprotective effect fora radiation therapy patient afflicted with a neoplastic disease, toeffectively treat a patient in need of radiation therapy, to supportbone marrow transplantation and to promote megakaryocyte production andmobilization and platelet production. In a more preferred embodiment,the concentration of active agent is between about 100 ng/kg body weightand about 10.0 mg/kg body weight. In a most preferred embodiment, theconcentration of active agent is between about 10 μg/kg body weight andabout 10.0 mg/kg body weight. This dosage regimen maximizes thetherapeutic benefits of the subject invention while minimizing theamount of agonist or peptide needed. Such an application minimizes costsas well as possible deleterious side effects.

For subcutaneous administration, the active ingredient may comprise from0.0001% to 10% w/w, e.g., from 1% to 2% by weight of the formulation,although it may comprise as much as 10% w/w, but preferably not morethan 5% w/w, and more preferably from 0.1% to 1% of the formulation.

In another preferred embodiment of the present invention, the activeagent is administered topically. Suitable topical doses and activeingredient concentration in the formulation are as described forsubcutaneous administration.

In a preferred embodiment of all of the aspects of the invention, theactive agent is selected from the group consisting of SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO:33, SEQ IDNO: 34; SEQ ID NO:35, SEQ ID NO:36; and SEQ ID NO:37.

In a further preferred embodiment of the above aspects of the invention,administration of the active agent is localized to the area affected bythe tissue-damaging radiation.

In another aspect of the present invention, an improved cell culturemedium is provided for megakaryocyte and platelet production, whereinthe improvement comprises addition to the cell culture medium of aneffective amount of between about 0.1 ng and 10 mg/ml of the activeagents of the invention. Any cell culture media that can supportmegakaryocyte and platelet production can be used with the presentinvention. Such cell culture media include, but are not limited to BasalMedia Eagle, Dulbecco's Modified Eagle Medium, Iscove's ModifiedDulbecco's Medium, McCoy's Medium, Minimum Essential Medium, F-10Nutrient Mixtures, Opti-MEM® Reduced-Serum Medium, RPMI Medium, andMacrophage-SFM Medium or combinations thereof.

The improved cell culture medium can be supplied in either aconcentrated (ie: 10×) or non-concentrated form, and may be supplied aseither a liquid, a powder, or a lyophilizate. The cell culture may beeither chemically defined, or may contain a serum supplement. Culturemedia is commercially available from many sources, such as GIBCO BRL(Gaithersburg, Md.) and Sigma (St. Louis, Mo.)

In a further aspect, the present invention provides kits formegakaryocyte and platelet production, wherein the kits comprise anamount effective for megakaryocyte and platelet production of the activeagents of the invention, and instructions for its use as a cell culturemedia supplement.

In a preferred embodiment, the kits further comprise cell culture growthmedium. Any cell culture media that can support megakaryocyte andplatelet production can be used with the present invention. Examples ofsuch cell culture media are described above. The cell culture medium canbe supplied in either a concentrated (ie: 10×) or non-concentrated form,and may be supplied as either a liquid, a powder, or a lyophilizate. Thecell culture may be either chemically defined, or may contain a serumsupplement.

In a further preferred embodiment, the kit further comprises a sterilecontainer, which can comprise either a sealed container, such as a cellculture flask, a roller bottle, or a centrifuge tube, or a non-sealedcontainer, such as a cell culture plate or microtiter plate (Nunc;Naperville, Ill.).

In another preferred embodiment, the kit further comprises an antibioticsupplement for inclusion in the reconstituted cell growth medium.Examples of appropriate antibiotic supplements include, but are notlimited to actimonycin D, Fungizone®, kanamycin, neomycin, nystatin,penicillin, streptomycin, or combinations thereof (GIBCO).

The present invention may be better understood with reference to theaccompanying example that is intended for purposes of illustration onlyand should not be construed to limit the scope of the invention, asdefined by the claims appended hereto.

EXAMPLE 1 Effect of AII on Rat Mortality and White Blood Cell Recoveryafter Irradiation

Female C57B1/6 mice (Jackson Labs, Bar Harbor, Me.) were irradiated with600 cGy total body irradiation. Subcutaneous injection with either AII(10 μg/kg/day or 100 μg/kg/day) or saline (placebo) was initiated twodays before (−day 2), on the day of (day 0) or 2 days after (+day 2)irradiation and continued until the animals succumbed to the irradiationor were necropsied. At various times after irradiation, the mice wereanaesthetized with Metofane (Pittman-Moore Animal Health, NZ) and bledvia the retro-orbital sinus. Red blood cells were lysed with 0.3% aceticacid and the number of white blood cells was determined by counting witha hemacytometer. The data in FIGS. 1-3 show that administration of AIIstarting at two days prior to irradiation did not protect againstmortality resulting from irradiation (FIG. 1), but that AIIadministration on the day of irradiation (FIG. 2) or two days afterirradiation (FIG. 3) substantially increased survival. Furthermore, AIIadministration at all time periods tested increased the number ofcirculating white blood cells (FIGS. 4-6). Further experimentsdemonstrated that AII administration increased the number ofmegakaryocytes (FIGS. 7-9), monocytes (FIGS. 10-12), neutrophils (FIGS.13-15), and lymphocytes (FIGS. 16-18). These data demonstrate that invivo administration of AII can improve hematopoietic recovery afterirradiation.

EXAMPLE 2 Effect of AII and AII Analogs/Fragments on WBC and PlateletNumbers After Irradiation

The animals were irradiated and treated as in Example 1, however,treatment started on day 0 only with one subcutaneous injection ofeither 10 μg/kg or 100 μg/kg daily until the study was terminated.Analogues and fragments of AII (see Table 3) were assessed for theireffect on WBC recovery and platelet number after irradiation. The dataare shown in FIGS. 20 and 21 and show that the peptides increase theproduction of both of these blood elements.

TABLE 3 Designation for Analogues/Fragments Name Abbreviation SequenceSEQ ID NO: GSD 28 Ile⁸-AII DRVYIHPI SEQ ID NO: 38 GSD 24B Pro³-AIIDRPYIHPF SEQ ID NO: 31 GSD 22A Ala⁴-AIII  RVYAHPF SEQ ID NO: 18 AII(1-7)DRVYIHP SEQ ID NO: 4 AII DRVYIHPF SEQ ID NO. 1

EXAMPLE 3 Effect of AII on Survival of Mice Receiving Bone MarrowTransplantation after Lethal Irradiation

Donor C57B1/6 mice (female, 6-8 weeks old) were irradiated with 600 cGytotal body irradiation. Starting on the day of irradiation, the micereceived either saline (0.1 ml) or 20 μg/ml angiotensin II (0.1 ml, 100μg/kg) subcutaneously for fourteen days. At the end of this period, thebone marrow was harvested from the femur by flushing and the number ofviable nucleated cells determined by counting under a light microscopeon a hemacytometer in the presence of trypan blue.

These donor bone marrow cells were then injected intravenously intorecipient mice (female C57B1/6, 6-8 weeks old) that had been lethallyirradiated (900 cGy total body irradiation) at two concentrations: 1×10⁶or 1×10⁵ cells per mouse. After injection, the recipient mice receivedeither saline or 100 μg/kg AII subcutaneously until death ortermination. The study design in its entirety is as follows:

Donor Recipient Cell Number Saline Saline 1 × 10⁶ Saline Saline 1 × 10⁵Saline All 1 × 10⁶ Saline All 1 × 10⁵ All Saline 1 × 10⁶ All Saline 1 ×10⁵ All All 1 × 10⁶ All All 1 × 10⁵

The survival of the mice and the number of circulating white blood cellswere measured as a function of time post-bone marrow transplantation.The data are presented in FIGS. 22-24, and demonstrate that AIItreatment increased both survival and white blood cell number in micereceiving bone marrow transplantation after irradiation. The greatestbenefit was conferred by treatment of both the donor bone marrow cellsand the recipient mice with AII.

The methods and kits of the present invention, by mitigating radiationinduced tissue damage and improving the efficacy of radiation therapy,significantly enhance the utility of presently available treatments bothfor radiation-induced tissue damage and for clinical radiation therapy,as well as bone marrow transplantation by increasing the survival rateof patients and accelerating the reconstitution of the patient'shematopoietic system. Similarly, by providing a method for megakaryocyteand platelet production, the present invention will greatly augmentclinical cancer treatments and bone marrow transplantation and otherconditions that lead to decreased megakaryocyte production andmobilization and platelet production.

The method of the present invention also increases the potential utilityof megakaryocytes as vehicles for gene therapy in hematopoieticdisorders, by providing a more efficient means to rapidly expandtransfected megakaryocytes.

It is to be understood that the invention is not to be limited to theexact details of operation, or to the exact compounds, compositions,methods, procedures or embodiments shown and described, as obviousmodifications and equivalents will be apparent to one skilled in theart, and the invention is therefore to be limited only by the full scopeof the appended claims.

1. An improved method of pluripotent stem cell transplantation, theimprovement comprising administering to a subject in need of pluripotentstem cell transplantation in amount effective of a peptide comprisingSEQ ID NO:4 to improve one or more of white blood cell survival, whiteblood cell number, subject survival, and platelet number in the subjectafter bone marrow transplantation, wherein the administering occurseither before, simultaneously with, or after pluripotent stem celltransplantation.
 2. The method of claim 1, wherein the subject undergoesradiation treatment prior to administration of the peptide.
 3. Themethod of claim 1, wherein the peptide is administered subcutaneously.4. The method of claim 1, wherein the peptide consists of SEQ ID NO:4.5. The method of claim 4, wherein the subject undergoes radiationtreatment prior to administration of the peptide.
 6. The method of claim4, wherein the peptide is administered subcutaneously.
 7. The method ofclaim 1, wherein the method further comprises contacting donorpluripotent stem cells with the amount effective of the peptide prior totransplantation of the donor bone marrow cells into the subject.
 8. Themethod of claim 1, wherein the amount effective of the peptide improveswhite blood cell survival in the subject after pluripotent stem celltransplantation.
 9. The method of claim 1, wherein the amount effectiveof the peptide improves white blood cell number in the subject afterpluripotent stem cell transplantation.
 10. The method of claim 1,wherein the amount effective of the peptide improves subject survivalafter pluripotent stem cell transplantation.
 11. The method of claim 1,wherein the amount effective of the peptide improves platelet numberafter pluripotent stem cell transplantation.
 12. The method of claim 4,wherein the amount effective of the peptide improves white blood cellsurvival in the subject after pluripotent stem cell transplantation. 13.The method of claim 4, wherein the amount effective of the peptideimproves white blood cell number in the subject after pluripotent stemcell transplantation.
 14. The method of claim 4, wherein the amounteffective of the peptide improves subject survival after pluripotentstem cell transplantation.
 15. The method of claim 4, wherein the amounteffective of the peptide improves platelet number after pluripotent stemcell transplantation.
 16. The method of claim 7, wherein the amounteffective of the peptide improves white blood cell survival in thesubject after pluripotent stem cell transplantation.
 17. The method ofclaim 7, wherein the amount effective of the peptide improves whiteblood cell number in the subject after pluripotent stem celltransplantation.
 18. The method of claim 7, wherein the amount effectiveof the peptide improves subject survival after pluripotent stem celltransplantation.
 19. The method of claim 7, wherein the amount effectiveof the peptide improves platelet number after pluripotent stem celltransplantation.
 20. The method of claim 4, wherein the method furthercomprises contacting donor pluripotent stem cells with the amounteffective of the peptide prior to transplantation of the donorpluripotent stem cells into the subject.
 21. The method of claim 20,wherein the amount effective of the peptide improves white blood cellsurvival in the subject after pluripotent stem cell transplantation. 22.The method of claim 20, wherein the amount effective of the peptideimproves white blood cell number in the subject after pluripotent stemcell transplantation.
 23. The method of claim 20, wherein the amounteffective of the peptide improves subject survival after pluripotentstem cell transplantation.
 24. The method of claim 20, wherein theamount effective of the peptide improves platelet number afterpluripotent stem cell transplantation.
 25. The method of claim 1,wherein the pluripotent stem cell transplantation comprises bone marrowtransplantation.
 26. The method of claim 4, wherein the pluripotent stemcell transplantation comprises bone marrow transplantation.
 27. Themethod of claim 5, wherein the pluripotent stem cell transplantationcomprises bone marrow transplantation.
 28. The method of claim 7,wherein the pluripotent stem cell transplantation comprises bone marrowtransplantation.
 29. The method of claim 12, wherein the pluripotentstem cell transplantation comprises bone marrow transplantation.
 30. Themethod of claim 13, wherein the pluripotent stem cell transplantationcomprises bone marrow transplantation.
 31. The method of claim 14,wherein the pluripotent stem cell transplantation comprises bone marrowtransplantation.
 32. The method of claim 20, wherein the pluripotentstem cell transplantation comprises bone marrow transplantation.
 33. Themethod of claim 21, wherein the pluripotent stem cell transplantationcomprises bone marrow transplantation.
 34. The method of claim 22,wherein the pluripotent stem cell transplantation comprises bone marrowtransplantation.
 35. The method of claim 23, wherein the pluripotentstem cell transplantation comprises bone marrow transplantation.
 36. Themethod of claim 24, wherein the pluripotent stem cell transplantationcomprises bone marrow transplantation.